Parasitic helminthes (worms) are a significant health and economic burden: over two billion people are infected by helminthes [1], and parasitic nematodes cause billions of dollars of crop damage each year in the United States [2]. The developmental stages of these organisms are widely studied [3, 4]. One stage, dauer (German for "duration", also known as an alternative L3 larval stage) covers an alternative larval stage in which development stops and the worms enter a hibernation-like state in which they can survive extremely harsh environmental conditions, often for years. In the case of parasitic nematodes, this resting state is quite often the infectious state [5]. As the burden of parasitic nematodes grows in the face of emerging resistance to the few existing antihelminthic agents, it is becoming increasingly important to understand the life cycles of parasitic worms so that new drugs may be developed [1]. The nuclear receptor DAF-12 (for "dauer formation"), first identified in C. elegans, is known to control many nematode species' entry into and exit from the dauer resting state [6]. Daf-12 belongs to a family of over 30 genes which transduce environmental signals to influence the choice between dauer or reproductive development. Favorable environments activate insulin/IGF and TGF-beta pathways converge, leading to production of the steroid hormone dafachronic acid (DA), which binds and activates Daf-12 [7]. Currently available antihelminthic agents, to which resistance is beginning to emerge, act primarily on the feeding stages of the worms and have little effect on the infectious stages [8]. Therefore, pharmacologic activators developed through high-throughput screening would be used both practically as nematicides and academically as tools to characterize the role of DAF-12 in modulating life cycle [8, 9].

Assay Overview:The purpose of this assay is to determine non-specific gene reporter activation of compounds found active in a set of experiment entitled "Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite S. stercoralis (ssDAF-12)" (AID 652126). Compounds are tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 68 uM.Protocol Summary:In this assay, HEK293 are transfected with a GAL4-responsive reporter plasmid (tk-luc) and expression vectors encoding Gal4-LXR. The ability of compounds to alter LXR-mediated transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. The LXR agonist T0901317 was used as a positive control for this assay.The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora) and placed in the incubator for 3 hours. Cells were then treated with 34 nL/well of either test compounds, DMSO (Low Control, final concentration 0.68%) or 6.8 uM of T0901317 (High Control). Plates were incubated for 24 hours at 37 C, 5% CO2 and 95%RH and then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).The percent activation of each test compound was calculated as follows:%_Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median__Low_Control ) )Where:High_Control is defined as wells treated with 6.8 uM T0901317 compoundLow_Control is defined as wells treated with DMSO only.Test_Compound is defined as wells treated with test compound.For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 68 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 68 uM.PubChem Activity Outcome and Score:Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.The PubChem Activity Score range for inactive compounds is 0-0, there are no active compounds.List of Reagents:MH100-tk-luc luciferase reporter plasmid (Assay Provider)Gal4-LXR expressing plasmid (Assay Provider)List of Consumables:HEK293 cells (ATCC, part CRL-1573)DMEM (Invitrogen, part 11965)FBS (Hyclone, part SH30088.03)Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)TransIT 293 (Mirus Corporation, part MIR-2700)OptiMEM (Invitrogen, part 31985)TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)One-Glo (Promega, part E6130)1536-well plates (Greiner part 789173)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate luciferase activity and hence well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.

inhibition Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.

String

2

IC50*

The concentration at which 50 percent of the inhibition in the inhibitor assay is observed; (IC50) shown in micromolar.

Float

μM

3

LogIC50

Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration

Float

4

Maximal Response

The maximal or asymptotic response above the baseline as concentration increases without bound.

Float

5

Baseline Response

Adjustable baseline of the curve fit, minimal response value.

Float

6

Inflection Point Concentration

The concentration value for the inflection point of the curve.

Float

μM

7

Hill Slope

The variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.

Float

8

Response Range

The range of Y.

Float

9

Chi Square

A measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.

Float

10

Rsquare

This statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.

Float

11

Number of DataPoints

Overall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.

Integer

12

Excluded Points

Flags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.